Abstract Background: The bone marrow harbors tumor-antigen specific T cells in patients with breast cancer (1). Marrow-infiltrating lymphocytes (MILs®) are the product of activating and expanding bone-marrow T cells (2). In multiple myeloma, transfer of MILs as adoptive cell therapy has demonstrated anti-tumor activity (3). The use of adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) has been used successfully for hormone-receptor positive (HR+) metastatic breast cancer (4). Expansion of MILs was previously shown to be feasible in breast cancer, non-small cell lung cancer (NSCLC), prostate, head and neck, glioblastoma and multiple myeloma (5). Using an expanded cohort, we demonstrate that tumor-specific MILs can be expanded from bone marrow in patients with breast cancer. Methods: Bone marrow aspiration and blood samples were collected from 8 patients with metastatic and high-risk early-stage breast cancer, including 3 patients with HR+/HER2-, 2 patients with HER2+, and 3 patients with triple negative subtypes, at Providence Cancer Institute (Portland, OR). MILs and peripheral blood lymphocytes were activated and expanded from patient samples during a 10-day proprietary process. T cell lineage-specific markers CD3, CD4 and CD8 were characterized by flow cytometry pre- and post- expansion. Tumor-specific T cells were quantitated in expanded MILs and peripheral blood lymphocytes using a previously described cytokine-secretion assay (3). Briefly, they were defined as the IFNy-producing population by flow cytometry. Autologous antigen-presenting cells (APCs) were pulsed with lysates from allogeneic cancer cell lines and co-cultured with activated MILs or peripheral blood lymphocytes. APCs pulsed with irrelevant mis-matched cancer cell line lysates or media alone were used as negative controls. Results: In all harvested samples, MILs expansion successfully resulted in a selective increase in CD3+ T cells. Cytokine-producing CD4+ and CD8+ T cells were detected in all expanded MILs samples, but not in any of the matched activated and expanded peripheral blood lymphocytes. MILs were successfully expanded in HR+/HER2-, HER2+, and triple negative subtypes without apparent differences in activity as measured by cytokine production. Conclusion: MILs isolated from this expanded cohort of metastatic and high-risk early stage breast cancer patients demonstrated cytokine production in response to pulsed cancer cell line lysates, consistent with our experience with other solid (5) and hematological (3) malignancies. A phase II trial to evaluate MILs in combination with a checkpoint inhibitor is underway in patients with anti-PD1/PDL1-refractory NSCLC (NCT04069936). These preclinical data demonstrate that expanding MILs is feasible and could be evaluated therapeutically in breast cancer.